A polyvalent DMA prime-protein boost vaccine (DP6-001), consisting of codon optimized HIV-1 env (A, B, C, E) and gag (C) genes and homologous gp120 proteins in QS-21 adjuvant, was evaluated by our team in both preclinical studies and in a Phase I clinical trial. Data from the clinical trial demonstrated that DNA priming enhanced the anti-Env antibody (Ab) response following Env protein boost (see Preliminary Studies). This was the first time that this vaccine strategy for eliciting Ab, termed 'DNA prime/protein boost,'was demonstrated in humans for HIV vaccine development. Especially encouraging was the detection of neutralizing antibodies (NAbs) against both homologous and heterologous primary isolates. In addition, T cell responses against Gag and Env were also induced by DP6-001 vaccination, indicating that the DNA prime-protein boost strategy has the potential to elicit both anti-HIV NAbs and cellular immunity. While these results were exciting, the human trial also revealed unanticipated reactogenic complications that were not seen in either the preclinical, IND-enabling safety/toxicology testing in rabbits or the nonhuman primate studies. A single case of leukocytoclastic vasculitis (LCV) that transiently occurred in a subject who received the highest DNA priming dose (7.2 mg intramuscularly) followed by a single protein immunization. Similarly, all of the other five subjects from this high dose DNA priming group also exhibited reactogenic AEs as evidenced by self-reported myalgias, low grade fever and headaches following the single protein boost. These results have made us aware of a boundary condition for an acceptable HIV vaccine: while strong immunogenicity is good, excessive reactogenicity could limit the widespread deployment of such a vaccine. As a result of this experience, we have reconfigured our studies in Project 2 in an effort to minimize reactogenicity as we optimize immunogenicity. Aim 1 will use mice to compare the cytokine profile and TLR responses between mice receiving low and high DNA prime when protein boost includes QS-21 which was included in DP6-001. Aim 2 will use the information learned from Aim 1 to test the effect of other adjuvants (monophosphoryl lipid A, Montanide ISA 51 and alum) that may have lower potential for reactogenicity while maintaining the high immunogenicity. Aim 3 will examine the reactogenicity of the candidate vaccines in mice when DNA vaccine is delivered by electroporation. Aim 4 will test the next generation of polyvalent Env formulation (including optimized adjuvant and DNA delivery method) in rhesus macaques to assess their immunogenicity, safety, and ability to protect from a viral challenge.